The present invention relates to a new process for the stereoselective preparation of substituted cyclohexylcyanohydrins.
It is known that cyanohydrins can be prepared by reaction of ketones with hydrocyanic acid (see, for example, Beyer, Walter, Lehrbuch der Organischen Chemie [Textbook of Organic Chemistry], 21st Edition, S. Hirzel Verlag Stuttgart 1988, page 213). In substituted cyclic ketones, however, this reaction does not proceed stereoselectively with sterically less demanding radicals.
Protected cyanohydrins are obtained in the reaction of ketones with trimethylsilyl cyanide (see the reference cited above, Beyer, Walter p. 535 ff., Hiyama, Saito, Synthesis 1985, 645-647). The use of oxynitrilases for the preparation of optically active cyanohydrins is also known (see, for example, EP-A-0 799 894).
It was the object of the present invention to make available a process for the more stereoselective preparation of substituted cyclohexylcyanohydrins.
The present invention relates to a process for the stereoselective preparation of cyclohexylcyanohydrins of the formula (I) 
in which
R represents alkyl, cycloalkyl (in which a methylene group is optionally replaced by oxygen), alkoxy, alkenyloxy, cycloalkyloxy, arylalkyloxy, aryloxy or aryl, in each case optionally substituted,
which is characterized in that cyclohexanones of the formula (II) 
in which
R has the abovementioned meanings,
are reacted with a cyanide source in the presence of an oxynitrilase and if appropriate in the presence of a diluent.
The wavy line in the formula (I) means that the compounds of the formula (I) are mixtures of cis and trans isomers of the following structure: 
Surprisingly, the compounds of the formula (I) are obtained in good yield. Reactions of cycloalkanones with oxynitrilases to give cyanohydrins were not known.
Furthermore, the process according to the invention proceeds more rapidly than the purely chemical process.
The process according to the invention furthermore surprisingly yields, depending on the oxynitrilase employed, an excess of desired isomers (cis or trans).
The process according to the invention can be represented by the following equation if hydrocyanic acid is used as a cyanide source: 
Formula (II) provides a general definition of the compounds needed as starting substances for the process according to the invention.
Preferred substituents or ranges of the radical R shown in the formulae mentioned above and below are explained below.
Compounds preferably employed in the process according to the invention are those of the formula (II) in which
R represents C1-C8-alkyl which is optionally substituted by halogen, C3-C8-cycloalkyl (in which one methylene group is optionally replaced by oxygen) which is optionally substituted by C1-C6-alkyl or C1-C6-alkoxy, C1-C8-alkoxy which is optionally substituted by halogen, C3-C6-alkenyloxy which is optionally substituted by halogen, C2-C8-cycloalkyloxy which is optionally substituted by C1-C6-alkyl or C1-C6-alkoxy, or phenyl, phenoxy or benzyloxy, each of which is in each case optionally substituted by halogen, C1-C6-alkyl, C1-C6-halogenoalkyl, C1-C6-alkoxy or C1-C6-halogenoalkoxy.
Compounds particularly preferably employed are those of the formula (II) in which
R represents C1-C6-alkyl which is optionally substituted by halogen, C3-C6-cycloalkyl (in which a methylene group is optionally replaced by oxygen) which is optionally substituted by C1-C4-alkyl or C1-C4-alkoxy, C1-C6-alkoxy which is optionally substituted by halogen, C3-C6-alkenyloxy which is optionally substituted by halogen, C3-C6-cycloalkyloxy which is optionally substituted by C1-C4-alkyl or C1-C4-alkoxy, or phenyl, phenoxy or benzyloxy, each of which is in each case optionally substituted by halogen, C1-C4-alkyl, C1-C2-halogenoalkyl, C1-C4-alkoxy or C1-C2-halogenoalkoxy.
Halogen in the above definitions represents fluorine, chlorine, bromine and iodine, particularly fluorine, chlorine and bromine, in particular fluorine and chlorine.
Compounds very particularly preferably employed are those of the formula (II) in which
R is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, tert-butyl, cyclopentyl, cyclohexyl, allyloxy, 2-butenyloxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, phenyl or benzyloxy.
In particular, the following compounds of the formula (II) may be mentioned:
The compounds of the formula (I) prepared according to the invention are known in some cases or can be prepared by the processes described in the literature cited at the beginning (M. Mousseron et. Al.; Bull. Soc. Chim. Fr. 4. 1435-39 (1970); P. Geneste et. al.; Bull. Soc. Chim. Fr., II, 187-191 (1980)).
The compounds of the formula (II) needed as starting substances for carrying out the process according to the invention are known in some cases or can be prepared by processes which are known in principle (compare Example II-1 and, for example, J. Org. Chem. 47 3881, 1982).
The process according to the invention is preferably carried out in the presence of a diluent which is inert to the reactants.
Aliphatic or aromatic hydrocarbons, such as benzine, toluene, xylene and tetralin, can preferably be used, in addition aliphatic or aromatic halogenohydrocarbons, such as methylene chloride, chloroform, carbon tetrachloride, chlorobenzene and o-dichlorobenzene, furthermore open-chain or cyclic ethers, such as diethyl ether, methyl tert-butyl ether, diisopropyl ether, tetrahydrofuran and dioxane, in addition carboxylic acid esters, such as ethyl acetate, and also strongly polar solvents, such as dimethyl sulphoxide and sulpholane, or alternatively alcohols such as methanol, ethanol, isopropanol and tert-butanol.
Preferred diluents are ethers (in particular dilsopropyl ether) and carboxylic acid esters (in particular ethyl acetate).
The reaction according to the invention can also be carried out in a two-phase system which consists of water and an organic solvent which is not completely miscible with water. For this, suitable organic solvents are, for example, methyl tert-butyl ether, diisopropyl ether and ethyl acetate. Diisopropyl ether is preferably used.
The process according to the invention can also be carried out in an aqueous system in the absence of an organic solvent at a preferred pH of  less than 5, in particular at a pH of between 3 and 4. The pH is adjusted by means of a buffer. Suitable buffers are, for example, sodium acetate or sodium citrate. Preferably, a 20 to 500 mM sodium citrate buffer is used.
The process according to the invention is carried out in the presence of an oxynitrilase (hydroxynitrile lyase). Oxynitrilases are enzymes which catalyse the cleavage and formation of cyanohydrins. Oxynitrilases have long been known (see, for example, EP-A-0 799 894; Chem. Commun. 1997, 1933; Angew. Chem. 1994, 106, 1609-1619; Angew. Chem. Int. Ed. Engl. 1996, 35, 438; Enantiomer, Vol. 1, pp. 359-363).
For the process according to the invention, the following oxynitrilases are preferably employed: (R)-oxynitrilase from the bitter almond (Prunus amygdalus), (S)-oxy-nitrilase from cassava (Manihot esculenta) and (S)-oxynitrilase from the rubber tree (Hevea brasiliensis). The (R)-oxynitrilase from the bitter almond (Prunus amygdalus) and the (S)-oxynitrilase from cassava (Manihot exculenta) are particularly preferably employed. The (R)-oxynitrilase from the bitter almond (Prunus amygdalus) is very particularly preferably used.
Depending on the type of oxynitrilase employed, the process according to the invention leads to an excess of trans or cis isomers. In some cases, a dependence of the reaction course on the type of substituent R is moreover to be observed.
The use of the (R)-oxynitriliase from bitter almond basically leads to an excess of trans isomers.
When using the (S)-oxynitrilase from cassava, the trans isomer surprisingly predominates for the small radicals R methyl and methoxy, while for larger radicals
R an excessively cis isomer is regularly obtained.
With smaller radicals R (for example methoxy), the use of the (S)-oxynitrilase from the rubber tree likewise yields an excess of trans isomers.
The obtainment of the oxynitrilases mentioned is described in the literature.
For the isolation of the (R)-oxynitrilase [EC 4.1.2.10] from bitter almonds see, for example, Biochem. Z. 1963, 337, 156-166 and 1966, 346, 301-321; Proteins 1994, 19, 343-347; Helv. Chim. Acta, 1983, 66, 489. For the isolation of the (S)-oxynitrilase [EC 4.1.2.37] from cassava see, for example, F. J. P. De C. Carvalho, Dissertation, University of California, Davis, 1981; Arch. Biochem. Biophys. 1994, 311, 496-502. For the isolation of the (S)-oxynitrilase from the rubber tree see, for example, Physiologia Plantarum 1989, 75, 97; Plant Science 1996, 115, 25-31.
Basically, the oxynitrilases can be employed in the process according to the invention in various preparation forms.
On the other hand, crude enzyme can also be used, i.e. the enzyme is not isolated, but a cell-free extract is employed which is obtained by disrupting the cells with ultrasound, centrifuging off the crude extract thus obtained from the cell walls/membranes and concentrating it. References, e.g. (R)-oxynitrilase: Tetrah. Lett. 1988, 29, 4485-88, Tetrah. Ass. 1996, 7, 1105-1116, Synthetic Communications 1991, 21, 1387-91.
For the use of (R)-oxynitriles from bitter almonds, the use of preferably defatted almond flour which is obtained by grinding bitter almonds and extracting them with, for example, hexane or petroleum ehter (commercially available from Sigma) or ref.: Lett., 1988, 29, 4485-4488 is suitable.
Purified enzyme is preferably used.
The enzymes can be bound to suitable carrier material for carrying out the reaction according to the invention. Suitable carrier materials are, for example, celluloses, in particular nitrocellulose and cellulose P 100 PSC: Elcema(copyright); Degussa, particle size 50-150 xcexcm, coated on the surface with 2% of amorphous silicic acid (Aerosil). After the reaction is complete, this procedure makes possible the removal of carrier-bound enzyme by filtration and thus the simplified work-up. The carrier-bound enzyme thus recovered can moreover be used again.
As a rule, the enzyme is applied to the carrier by adding the enzyme solution dropwise to the carrier, see also, for example, Angew. Chem. Int. Ed. Engl. 1996, 35, 439.
The process according to the invention is carried out in the presence of a cyanide source.
Suitable cyanide sources are all compounds which release cyanide ions (CNxe2x88x92). Examples which may be mentioned are hydrocyanic acid, cyanohydrins such as acetone cyanohydrin and metal cyanides such as KCN. Hydrocyanic acid or KCN, particularly preferably hydrocyanic acid, is preferably used.
The temperature in the process according to the invention can be varied within a relatively large range. In general, the reaction is carried out at temperatures between xe2x88x9220xc2x0 C. and 80xc2x0 C., preferably between 0xc2x0 C. and 40xc2x0 C.
The process according to the invention is in general carried out under normal pressure.
The molar ratio of compound of the formula (II) to cyanide ions is in general between 1:1 and 1:50, preferably between 1:1 and 1:4.
In general, when carrying out the process according to the invention, the compound of the formula (II) and the cyanide source are introduced into a suitable diluent, the immobilized enzyme is added and the reaction mixture is stirred at room temperature until the reaction is complete. The catalyst is then filtered off and the filtrate is worked up using customary methods.
In principle, it is also possible to carry out the reaction continuously, for example, by passing the starting materials dissolved in the diluent through a column which is loaded with the carrier-immobilized enzyme or allowing them to react in a membrane reactor, the products being separated from the starting materials and the catalyst by evaporation.
The compounds of the formula (I) prepared using the process according to the invention are valuable intermediates for the preparation of herbicides and pesticides according to the following reaction scheme: (see, for example, EP 0 647 637). 